State of the art

Since the pioneering work of Maarten Koornneef who identified a number of FTi
mutants in Arabidopsis thaliana this species has been used as a model
for genetic studies of FTi regulation. Many key regulators of FTi control have
been cloned and characterized in the past 15 years2. Scientists in
Germany have been at the forefront of FTi research.In the following we
give a short overview of the key genes and mechanisms that have been described
in Arabidopsis and other models and compare these with the current data
from crop species.

Unraveling the mechanisms of flowering time control in model and crop
species

Arabidopsis is an annual species which flowers in the first year
without extended exposure to cold temperatures. Biennials like sugar beet or
cabbage, in contrast, have an obligaterequirement for cold exposure
over winter for achieving floral competence in a process called vernalization.
Perennials like fodder grasses and trees often need many years to flower. It is
of utmost importance for this project that key regulators and pathways have been
found to be highly conserved across species borders. However, important
differences exist. In some cases important crop flowering time genes are absent
in the model Arabidopsis (e.g.Ghd7 of rice or
VRN2 of wheat). In other cases the same gene confers a different
response to environment, for example CO of Arabidopsis
promotes flowering in response to long summer days, whereas in rice its
orthologue represses flowering in long days. In general, changes in FTi
behaviour, e.g. winter and spring types, can be due to sequence changes within
individual FTi regulators.

Flowering genes are involved in the initial establishment of a
flowering-competent state, e.g. in the process of vernalization, and in
floral transition when meristems switch to reproductive growth and develop
flowers. Mutant analyses under various environmental conditions and genetic and
molecular interaction studies showed that several distinct regulatory pathways
exist. These pathways are generally referred to by the exogenous or endogenous
cues that they respond to, i.e. the vernalization, photoperiod,
gibberellin, and autonomous pathway. These pathways converge to regulate a set
of 'floral integrator' genes that integrate the outputs of the various pathways
and, under favorable conditions, directly activate floral meristem identity
genes (Figure 1). Subsequently, the vegetative shoot apical
meristem is turned into a floral meristem that initiates one or several flower
primordia.

Figure 1: Simplified scheme of genetic, endogenous (blue) and environmental (red)
factors for flowering time control in A. thaliana and implications on
plant production.

Conservation and divergence of vernalization pathways

Plants in temperate climates have evolved a signal perception and
transduction pathway that senses prolonged periods of cold over winter and
translates this environmental cue into an increased competence to flower, a
process known as vernalization. This process, often combined with a day
length-sensing mechanism, ensures flower development and flowering under
favorable conditions in spring or summer.

Vernalization requirement and response possess two intriguing features: a)
The temporal separation between the plant's exposure to cold in winter and the
onset of flowering in spring or summer, and b), the renewed vernalization
requirement for flowering in subsequent generations. Work in
Arabidopsis has revealed the epigenetic nature of the underlying
processes and established at the molecular level how the removal of a block to
flowering by vernalization enables the plant to respond to the second major
seasonal stimulus, daylength. One of the key genes in Arabidopsis that
regulate vernalization requirement and response is FLC, a MADS-box
transcription factor that acts as a repressor of floral transition. The
expression of FLC itself is tightly controlled by a plethora of both
positive and negative regulators (Figure 1). The response to
vernalization is facilitated by a cascade of gene regulatory processes and
results in chromatin-based and mitotically stable repression of FLC. In
the following generation, FLC expression is reset around the time of
early embryogenesis, thus ensuring a renewed requirement for vernalization.

While FLC orthologs in rapeseed and other Brassica crops
are clearly functionally related to FLC the extent of conservation of
FLC and the vernalization pathway as it has been described for
Arabidopsis outside the Brassicaceae is still controversial.
FLC-like ESTs were identified in the three major core eudicot clades
(rosids, asterids, and caryophyllids), but evidence for functional conservation
of the corresponding genes is scarce3. A notable exception is the
BvFL1 gene in sugar beet, a caryophyllid, which was shown to act as a
repressor of flowering when transformed into an ArabidopsisFLC null mutant.

Among monocots, vernalization requirement and response is best understood in
temperate cereals. Identification of the key regulators of vernalization
requirement in wheat, VRN1, VRN2 and VRN3, revealed a
regulatory pathway whose components differ from those of the
FLC-dependent vernalization pathway but are homologous to or share
conserved domains with other floral regulatory genes in Arabidopsis.
Recent data further indicate that the changes in expression of VRN1 in
barley during vernalization and maintenance of its state of transcriptional
activity post-vernalization involve chromatin modification. Thus, both
Arabidopsis and the cereals use an epigenetic mechanism to bridge the
temporal separation between induction of a flowering-competent state by
vernalization and initiation of flowering in the spring, although the known
targets, ArabidopsisFLC and wheat VRN1, are not
orthologous and have opposite effects on floral transition. Despite some
similarities in terms of protein domain organization and epigenetic regulation,
the stark differences in vernalization pathways in Arabidopsis and
temperate cereals suggests that vernalization requirement and response evolved
independently in the dicot and monocot lineages. This creates a need for further
research to unravel the FTi regulation in crop species.

Photoperiod and circadian clock control of flowering time

The extensive variation in seasonal temperature changes during the evolution
of flowering plants and the divergence of vernalization pathways (s. above)
stands in contrast to the stable annual photoperiodic conditions during the
history of the earth, which is consistent with an apparently higher degree of
functional conservation of photoperiodic control of flowering across taxa.

Photoperiod and seasonal changes in day length is a second environmental cue
with major effects on the timing of flowering during the course of a year. In
the (facultative) long-day plant Arabidopsis, a key role in the
regulatory pathway involved is played by the plant-specific CCT domain
transcription factor CO (CONSTANS). CO is regulated at the
transcriptional level by the circadian clock. Photoperiod control of floral
transition through CO and homologous genes is widely conserved among
flowering plants. Supporting evidence includes the identification of
CO-like genes from many monocot and dicot species4, and
complementation of the co mutation in Arabidopsis by CO-like
genes from species as distantly related to Arabidopsis as sugar beet or green algae5. However, the wide latitudinal variation between
photoperiodic conditions and the migration histories of plant species are likely
factors that contributed to the evolution also of differences in photoperiodic
response mechanisms among flowering plants. Thus, although the
CO-dependent promotion of flowering by activation of FT or its
orthologs appears to be conserved among long-day plants, short-day plants and
day-neutral plants differ from this scheme.

Interestingly, previous studies had shown that FLC and some of its
regulators modulate the period of the circadian clock. Together with recent
results on the regulation of the FLC-interacting protein SVP, these data may
suggest a regulatory loop or feedback interactions involving circadian clock
genes and FLC and SVP. Although on the basis of the current
evidence the regulatory interactions would appear relatively weak, they may
allow the fine-tuning of the plant's response to more subtle environmental
changes such as changes in ambient temperature.

Integration of different pathways and environmental factors to one
output

In Arabidopsis, the inputs from the vernalization and photoperiod
pathways are integrated by floral integrator genes that include the MADS-box
gene SOC1, FT and the FT homolog TSF, which
are strong promoters of flowering. FLC represses FT in leaves
and thus prevents production of FT protein, the long searched mobile signal
‘florigen’ that moves to the shoot apex and promotes flowering by activation of
AP14. In effect, the floral integrators are fully expressed
only after elimination of FLC repression as a result of vernalization,
and activation under long-day conditions by the photoperiod pathway. Like
CO, FT and other floral integrator genes are largely conserved
between dicots and monocots.

Clearly, other genetic factors than those discussed above also contribute to
the regulation of floral transition. The effect of changing ambient temperatures
on FTi is thought to be mediated by several genes, at least some of which are
also regulated by other floral regulatory pathways and thus may contribute to
the integration of different environmental signals. FTi is also affected by
various environmental stresses such as drought and heat. The plant’s response to
these stresses may be fine-tuned, at least in part, by differential interaction
of CO with a HAP-like protein complex6.

Flowering time regulation in perennials

The flowering behavior differs significantly between perennials and
annuals/biennials. Annuals and biennials go through senescence and die after
flowering. In contrast, perennials live for many years and flower repeatedly.
Perennials produce reproductive and vegetative meristems during one growth
season. While the floral meristems terminate their growth by developing flowers,
the vegetative meristems continue to grow during the subsequent season. Floral
transition in meristems which stayed vegetative in one growth season is
initiated in the following year after perception and transduction of seasonal
floral promoting signals such as cold or specific photoperiods. Evidently,
pathways linked to vernalization and photoperiod not only regulate flowering but
also regulate seasonal growth cessation and release from dormancy in perennial
plants.

Arabis alpina has been used as a model plant for perennials and
first studies have shown that key genes are differentially regulated compared to
annual Arabidopsis to confer characteristic patterns of perennial development7. Vice versa, downregulation of flowering genes in
Arabidopsis resulted in phenotypes common to perennial plants
suggesting only small molecular differences between perennials and annuals.
Flowering time QTL were located in perennial and annual forage grasses (L.
perenne, L. multiflorum) but no genes have been identified so far.
Other studies with woody perennials identified genes involved in seasonal floral
transition (e.g. strawberry, apple)8. Research is needed to
find the links between floral inducing signals and genetic factors to control
floral transition in woody perennials to tackle the problem of alternate
flowering/fruit bearing in fruit trees.

Annual and perennial plants enter a juvenile stage after germination. Plants
are unable to respond to flowering inducing signals during this stage.
Juvenility is species-specific and can be very short or last several years as it
is observed e.g. for trees. Overexpression of some floral promoting
genes or suppression of floral repressor genes have broken or at least shortened
the juvenile phase of trees like citrus, poplar or apple8. Also
epigenetic mechanisms seem to have an impact on the juvenile-to-adult vegetative
switch. However, factors that maintain the juvenile stage and thus repress
flowering are largely unknown and need to be investigated in the future.

Plant hormones

A direct influence of plant hormones in controlling flowering time sets in
with the initiation of floral meristems. In dependence of its concentration and
timing, auxin acts as a trigger stimulating the initiation of the inflorescence
meristem. Auxin might either derive from local auxin biosynthesis, or it might
be provided through the polar auxin transport pathway originating from the shoot
apical meristem9. On the other hand, differentiation of an axillary
meristem into a floral meristem is suppressed by cytokinins, which balance
between meristem differentiation and maintenance and thereby positively regulate
meristem size. While a contribution of root-to-shoot translocation of cytokinins
in maintaining meristem size is so far just weakly supported by physiological
studies, the essentiality of local cytokinin biosynthesis within the meristem
has been demonstrated in rice by mutations in the LOG gene, encoding an
enzyme involved in the last step of cytokinin biosynthesis or in CKX
encoding a cytokinin oxidase required for cytokinin degradation10.
More recently, transcriptome analyses of differentiating inflorescence meristems
also indicated an involvement of abscisic acid and jasmonic acid, which might
provide a regulatory link to the strong influence of abiotic stress factors on
flowering time (see “Flowering time and the adaptation to changing environmental
conditions”).

The role for gibberellins (GAs) in floral transition has so far been
characterized at a lower resolution. It is well established that under long-day
growth conditions or in biennial plants gibberellins mediate a photoperiodic
stimulus to flowering that relies on an upregulation of GA20-oxidase gene
expression in leaves. Evidence for GAs themselves representing this stimulus has
so far only been obtained in Lolium. Under short-day conditions,
however, the contribution of GA-dependent regulatory pathways even increases and
becomes obligatory. For instance in Arabidopsis, GAs promote flowering
through the activation of genes encoding the floral integrators SOC1, LFY and FT
in the inflorescence and floral meristems11.

Flowering time and the adaptation to changing environmental conditions

Sequence variations among FTi genes are important for the adaptation to
changing environmental conditions, both natural and artificial. Plants from
northern geographical regions germinate late and remain in the vegetative phase
over winter whereas plants flower early under arid climate conditions to avoid
drought stress. There is increasing evidence for a rapid evolution of FTi in
response to a climate fluctuation, e.g. by shifting the onset of
flowering to earlier dates12.

The great impact of FTi genes for the adaptation of crops to local
environments and production methods has been demonstrated. In seed crops,
flowering should be as early as possible to extend the seed filling phase, to
avoid harsh environmental conditions which endanger seed production or harvest
(e.g. drought, heat, frost), or to escape pathogen attack. By contrast,
delayed flowering may be desirable to realize high yields in biomass for energy
production. A number of flowering time QTL were located in association studies
using natural exotic and elite populations, e.g. wheat, barley13, rice, maize, rapeseed. In cereals, where natural allelic
variation was exploited from early on during domestication and selective
breeding for FTi traits (as co-determinants of yield) led to the prevalence of
different alleles in different growing areas, genetic mapping and association
studies with landraces and breeding material readily identified key loci with
major effects on FTi (e.g.VRN1 to -3, Ppd1).

Selection for FTi traits in the past was based exclusively on phenotypic
characteristics and relied on natural variation present in the primary and
secondary gene pool of a crop species. In the future FTi genes can be used by
breeders as functional markers for selecting favorable genotypes, for quality
control of seed lots, or for targeted manipulation of flowering traits by
genetic modification.

At first glance the application of FTi markers appears to be of limited value
because the onset of flowering is easy to score. Therefore, FTi markers will
hardly be used for routine selection. However, marker selection may be superior
over phenotypic selection after crossing adapted elite parents with non-adapted
(exotic) parents with inadequate flowering behavior. If this is due to
individual FTi genes, favorable plants can be easily identified in early
generations by a marker test whereas phenotypic selection on single plants can
be problematic due to environmental interaction.

Future work is needed in at least four areas: first, elucidation of how much
functional allelic variation is present for these major genes in wild and
domesticated cereal germplasm. Second, identification of new FTi regulators and
their interaction with internal and external factors. Third, analyses of how
these major genes interact both with other key players and with the environment
in crop species compared to model species like Arabidopsis, rice and
Brachypodium. And fourth, to find out, if newly characterized
functional alleles can be used in plant breeding to re-adjust the traits flower
initiation, flowering time and flowering duration to future breeding goals in
the light of changing climate conditions and agricultural practices.

Pleiotropic effects of flowering time genes

Yield potential, plant height and heading date are three classes of traits
that determine the productivity of many crop plants. Of immediate relevance to
yield potential, and therefore of great interest in plant breeding, is hybrid
vigor or heterosis. A recently finished SPP was dealing with heterosis in plants
and its genetic and metabolic reasons. There is increasing evidence that FTi
regulators and genes showing functional similarity are key players in
establishing heterosis in plants. Recently it was demonstrated that growth vigor
in hybrids of A. thaliana and A. arenosa was caused by
repression of the circadian clock genes CCA1 and LHY14. These findings may have some impact on future
heterosis research. The rice gene Ghd7 was cloned from a major QTL for
hybrid yield present in many Chinese rice hybrid cultivars15. This
gene encodes a CCT domain, typical for CO and related proteins. It has major
effects on an array of traits in rice, including number of grains per panicle,
plant height and heading date. Likewise, a homolog of FT was found to
account for heterosis in tomato16. Comparative QTL analysis of
heterosis in Arabidopsis and oilseed rape suggested that key flowering time loci
may coincide with significant QTL hotspots involved in regulation of biomass,
metabolite levels and seed yield. These results open new horizons for breeding
research because they suggest that FTi gene expression might trigger a cascade
of regulatory effects with a broad global effect on plant development and
yield.

Novel strategies by altering flowering time regulation

Genetic variation for FTi beyond the natural variation can be increased by
mutagenesis and transformation. Two or more mutations at different FTi loci can
be combined by crossing, and plants with novel FTi behavior can be selected. For
example, assuming that FTi repressors can behave in an additive or synergistic
manner, the hypothesis shall be tested that crossing two mutant plants with
delayed flowering phenotypes will produce a non-flowering hybrid. If confirmed,
this strategy will be followed to develop prototypes for breeding biennials and
perennials with novel FTi characters2.

Novel FTi characteristics can be also generated through targeted genetic
modification by transformation. The current knowledge of FTi control has been
exploited through either overexpression or suppression of gene activity2. Several
papers have been published describing transgenic plants with either up- or
down-regulation of FTi genes. In many cases the phenotypic effects were as
desired resulting in earlier or delayed flowering or even complete avoidance of
flowering 2. This demonstrates that manipulation of single genes can
have drastic effects on FTi and, as demonstrated above, other agronomic
characters in crop plants. The forage grass of the future should produce
non-flowering or reduced culms because leaf blades are more digestible and of
higher feeding value than sheaths and culms. Likewise, the future beet should be
planted before winter which requires full FTi control.

Literature Cited

2. Jung,C. and Müller,A. (2009) Flowering time control and applications in plant breeding. Trends in Plant Science 14, 563-573

16. Lifschitz,E. et al. (2006) The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proceedings of the National Academy of Sciences of the United States of America 103, 6398-6403